The present invention relates to novel proteins that form a complex with the type E botulin neurotoxin.
Various strains of the bacterium Clostridium, including C. botulinum, C. baratii, and C. butyricum, synthesize different serotypes of the potent neurotoxin botulin, which causes a form of food poisoning known as botulism. C. botulinum synthesizes seven different serotypes of botulin, which are designated by the letters A through G. Humans and other animals come into contact with these neurotoxins most frequently by consuming food that is improperly stored in a way that permits growth of anaerobic bacteria. Foods that are typically tainted with botulin include low acid canned meats and vegetables, preserved meats and fish, and pasteurized processed cheese spreads (Fogeding, In Foodborne Microorganisms and Toxins: Developing Methodology, M. D. Pierson and N. Sterns, Eds., Marcel Dekker, Inc., New York, N.Y., 1986; Kautter et al, J. Food Prot. 42:784-786, 1979).
Another form of botulism, infant botulism, is thought to be caused by consumption of ubiquitous spores of C. botulinum along with food (Simpson, In Botulinum Neurotoxin and Tetanus Toxin, Academic Press, San Diego, Calif., 1989). These spores may colonize the infant intestine, germinate, and produce the neurotoxin. Similarly, spores that gain access to deeply wounded tissue may germinate and produce neurotoxin within the wound.
Once present in the body, botulin neurotoxins cause muscle paralysis by blocking the release of acetylcholine from cholinergic nerve endings (DasGupta et al., Biochemistry and Pharmacology of Botulinum and Tetanus Neurotoxins, In Perspective in Toxicology, A. W. Bernheimer, Ed., Wiley, New York, N.Y., 1977). Death may be caused by paralysis of the respiratory muscles.
The nucleotide sequences of the genes encoding all of the different serotypes of the neurotoxin are known (Binz et al., J. Biol. Chem. 265:9153-9158, 1990; Campbell et al., J. Clin. Microbiol. 31:2255-2262, 1993; East et al., FEMS Microbiol. Lett. 96:225-230, 1992; Hauser et al., Nucl. Acids Res. 18:4924, 1990; Whelan et al., Eur. J. Biochem., 204:657-667, 1992; and Whelan et al., Appl. Environ. Microbiol. 58:2345-2354, 1992). These genes are coordinately regulated with those encoding proteins that form complexes with the various serotypes of botulin (Fujii et al., J. Gen. Microbiol. 139:79-83, 1993; and Nukina et al., In Botulinum and Tetanus Neurotoxins, B. R. DasGupta, Ed., Plenum Press, New York, N.Y., 1993). The A and B type neurotoxins are associated with at least five other proteins, called “neurotoxin binding proteins” or NAPS, while the type E neurotoxin has been found in association with only one other protein (Sugii et al, Infect. Immunol. 12:1262-1270, 1975; Sakaguchi, Pharmac. Ther. 19:165-194, 1983; Schantz et al., Microbiol. Rev. 56:80-99, 1992; and Singh et al., J. Protein Chem. 14:7-18, 1995).
The proteins that associate with the type A neurotoxin play a critical role in the food poisoning process by protecting the neurotoxin from the acids and proteolytic enzymes present in the gastrointestinal tract. For example, it is known that the oral toxicity of the intact type A neurotoxin complex is 43,000 times greater than the oral toxicity of isolated and purified type A neurotoxin (Sakaguchi, Pharmac. Ther. 19:165-194, 1983). The proteins associated with other serotypes similarly “protect” the neurotoxin, but to a lesser degree.